In many developing tissues, the generation of distinct cell types is initiated by the action of extracellular signals provided by local organizing centers. Certain signals have the additional feature of directing distinct cell fates at different threshold concentrations, and thus function as morphogens (Wolpert, 1969). In Drosophila, the patterning of embryonic segments and imaginal discs involves the graded signaling activities of the Hedgehog, Wingless and TGFβ-related proteins (Lawrence and Struhl, 1996). In vertebrate embryos the specification of mesodermal cell types has similarly been suggested to depend on the graded signaling activity of members of the TGFβ family (Smith, 1995; McDowell and Gurdon, 1999). The generation of cell pattern through morphogen signaling demands an effective means of converting graded extracellular activities into all-or-none distinctions in cell fate. But the mechanisms used to achieve such conversions have been poorly defined, particularly in vertebrate tissues.
In the developing vertebrate nervous system, Sonic hedgehog (Shh) appears to function as a gradient signal. The secretion of Shh by the notochord and floor plate controls the specification of ventral cell types (Marti et al., 1995; Roelink et al., 1995; Chiang et al., 1996; Ericson et al., 1996). Five distinct classes of ventral neurons can be generated in vitro in response to progressive two-to-three fold changes in extracellular Shh concentration (Ericson et al., 1997a, b). Moreover, the position at which each of these neuronal classes is generated in vivo is predicted by the concentration of Shh required for their induction in vitro: neurons generated in progressively more ventral regions of the neural tube require correspondingly higher concentrations of Shh for their induction (Ericson et al., 1997a). These observations have led to the view that the position that ventral progenitor cells occupy within a ventral-to-dorsal gradient of extracellular Shh activity directs their differentiation into specific neuronal subtypes (Ericson et al., 1997b).
In turn, these findings have focused attention on the steps by which graded Shh signaling directs the diversification of neural progenitor cells. Several homeodomain proteins, Pax7, Pax3, Pax6, Dbx1, Dbx2 and Nkx2.2, are expressed by ventral progenitor cells and their expression is regulated by Shh signaling (Goulding et al., 1993; Ericson et al., 1996; Ericson et al., 1997a; Briscoe et al., 1999; Pierani et al., 1999). Moreover, the pattern of generation of certain ventral neuronal subtypes is perturbed in mice carrying mutations in these Pax genes and in the Nkx2.2 gene (Ericson et al., 1997a; Mansouri and Gruss, 1998; (Briscoe et al., 1999), supporting the view that homeodomain proteins expressed by ventral progenitor cells regulate neuronal subtype identity. However, two important aspects of the link between Shh signaling and neuronal identity remain obscure. First, it is unclear how the presumed extracellular gradient of Shh activity results in stable and sharply delineated domains of homeodomain protein expression within ventral progenitor cells. Second, the spatial information provided by the homeodomain proteins characterized to date is insufficient to explain the diversity of neuronal subtypes generated at different dorsoventral positions.
In the first series of experiments these two issues are addressed. It is show first that the homeodomain proteins Nkx6.1 and Irx3 are expressed by progenitor cells in discrete domains of the ventral neural tube and are regulated by graded Shh signaling. The differential expression of five class I (Shh-repressed) proteins, Pax7, Irx3, Dbx1, Dbx2 and Pax6, and two class II (Shh-induced) proteins, Nkx6.1 and Nkx2.2, subdivides the ventral neural tube into five cardinal progenitor domains. Misexpression of individual proteins in the neural tube in vivo in these experiments provides evidence that cross-repressive interactions between class I and class II proteins establish individual progenitor domains and maintain their sharp boundaries, suggesting a mechanism by which graded Shh signals are converted into all-or-none distinctions in progenitor cell identity. In addition, the experiments show that the spatial patterns of expression of Nkx6.1, Irx3 and Nkx2.2 are sufficient to direct both the position and fate of three neuronal subtypes generated in ventral third of the neural tube. These findings suggest a model of ventral neuronal patterning that may provide insight into how extracellular signals are interpreted during the patterning of other vertebrate tissues.
Distinct classes of neurons are generated at defined positions in the ventral neural tube in response to a gradient of Sonic Hedgehog (Shh) activity. A set of homeodomain transcription factors expressed by neural progenitors act as intermediaries in Shh-dependent neural patterning. These homeodomain factors fall into two classes: class I proteins are repressed by Shh and class II proteins require Shh signaling for their expression. The profile of class I and class II protein expression defines five progenitor domains, each of which generates a distinct class of post-mitotic neurons. Cross-repressive interactions between class I and class II proteins appear to refine and maintain these progenitor domains. The combinatorial expression of three of these proteins—Nkx6.1, Nkx2.2 and Irx3—specifies the identity of three classes of neurons generated in the ventral third of the neural tube.
Sonic hedgehog (Shh) signaling has a critical role in the control of neuronal fate in the ventral half of the vertebrate central nervous system (CNS). The genetic programs activated in Shh-responsive progenitor cells, however, remain poorly defined. To test whether members of the Nkx class of homeobox genes have a prominent role in the specification of ventral cell types the second series of experiments examined patterns of neurogenesis in mice carrying a targeted mutation in the Nkx class homeobox gene Nkx6.1. In Nkx6.1 mutants there is a dorsal-to-ventral switch in the identity of progenitor cells and in the fate of post-mitotic neurons. At many axial levels there is a complete block in the generation of V2 interneurons and motor neurons and a compensatory ventral expansion in the domain of generation of V1 neurons. These studies support the idea that an Nkx gene code controls regional pattern and neuronal fate in the ventral region of the mammalian CNS.